Project Summary/Abstract Volatile anesthetics are essential to modern medicine, but despite their widespread clinical use, their precise cellular and molecular mechanisms of action remain unclear. Volatile anesthetics, such as isoflurane, depress synaptic transmission with both pre- and post-synaptic effects including inhibition of activity-dependent Ca2+ influx into the presynaptic nerve terminal. However, the principal presynaptic sites of action upstream of Ca2+ entry are unknown. Axonal endoplasmic reticulum (ER) Ca2+ controls presynaptic Ca2+ through ER Ca2+ sensing proteins, and decreased ER Ca2+ has been linked to a reduction in presynaptic Ca2+ influx through these proteins. ER Ca2+ efflux and influx mechanisms are essential for Ca2+ regulation and provide possible targets for anesthetic action. For example, in skeletal muscle, mutations in sarcoplasmic reticulum (SR) Ca2+ efflux receptors: ryanodine receptors (RyR) result in patient with episodes of malignant hyperthermia (MH) after treatment with volatile anesthetics. MH is a potentially fatal pharmacogenetic disorder characterized by hyperthermia, tachycardia, muscle rigidity, and hypermetabolism [1]. However, MH's effect on neurons is unknown. As my preliminary data show, isoflurane decreases activity-dependent ER Ca2+ surges. Isoflurane inhibits presynaptic Ca2+ entry and synaptic vesicle (SV) exocytosis, which I hypothesize involves effects on ER Ca2+ efflux dynamics through ryanodine receptors (RyR). I will address this hypothesis through the following specific aims: 1) Determine the effects of isoflurane on ER Ca2+ efflux and influx pathways; 2) Determine the effect of isoflurane on ER Ca2+ in neurons from an MH model mouse. Primary cultures of rat hippocampal neurons will be used to test isoflurane-induced changes in ER Ca2+ concentration using fluorescent biosensors and pharmacological modulators of ER Ca2+ regulators. Under the mentorship of Dr. Hugh C. Hemmings Jr. at Weill Cornell Medicine, I plan to further our understanding of isoflurane's neuronal mechanisms of action. Successful completion of these aims will reveal novel presynaptic mechanisms and aid in the development of more-selective anesthetics decreasing patient risk as well as facilitate the improvement of MH patient outcomes.